鼠抗人TCR-CD3单克隆抗体的制备及其初步应用.DOCVIP

临床医学论文-鼠抗人TCR/CD3单克隆抗体的制备及其初步应用 ??????????????? 作者：陈宏远,芮雯,花文峰，杜军,蔡绍晖 【摘要】? 目的 应用B淋巴细胞杂交瘤技术，制备鼠抗人CD3 单克隆抗体，并用于T淋巴细胞亚类检测。方法 以本室纯化的CD3蛋白免疫Balb/c小鼠，取免疫小鼠脾细胞与SP2/0骨髓瘤细胞融合，成功地获得1株抗人CD3单克隆抗体。经间接ELISA测定，杂交瘤细胞培养腹水的抗体效价。结合SDSPAGE实验考察单克隆抗体对CD3促T淋巴细胞增殖活性的作用。结果 杂交瘤细胞培养腹水的抗体含量为20 mg/mL，效价为10-7，并对T淋巴细胞有促增殖活性，检测正常人外周血阳性T细胞百分率为(73±7)％。结论 制备的抗人CD3单克隆抗体具备较高的纯度和活性，可用于T细胞亚类的检测。 【关键词】? CD3；单克隆抗体；杂交瘤 ??? Abstract: Objective To detect the CD3 positive T lymphocytes subgroup with the monoclonal antibody of mouse antihuman CD3 antigen of T lymphocyte which produced by B lymphocyte hybridoma technique. Methods Balb/c mice were immunized with CD3 protein produced in our laboratory, mixed SP2/0 myeloma with spleen cells of the mice, then obtained an antihuman CD3 antigen of hybridoma cell line. The ascitic culture monoclonal antibody titer was determined by indirectELISA, the quantity and molecular weight were determined by SDSPAGE. MTT method was contributed to explore its activity of promoting T lymphocyte proliferation. Results The monoclonal antibody quantity of ascitic hybridoma cell was 20 mg/mL and the titer was 10-7. It had an activity of promoting T lymphocyte proliferation, percentage of T lymphocyte in healthy adult peripheral blood samples was (73±7)％.Conclusion This results suggest that the antiCD3 monoclonal antibody had high purity and activity that might be used in the detection of? CD3? positive T lymphocytes subgroup, and could be applied in T lymphocyte detection. ??? Key words: CD3;monoclonal antibody; hybridomas ?? CD3是T淋巴细胞的一个分化抗原，表达于所有成熟T细胞表面，它是由5条肽链非共价结合组成的复合分子，分别称为γ、δ、ε、ζ和η链。其与TCR分子以非共价结合形成一个TCR/CD3复合受体分子，其中TCR是识别异种抗原和自身MHC分子多态性决定族的受体，而CD3分子并不参与抗原识别，它具有稳定TCR结构和传递活化信号的作用。TCR/CD3复合体是T细胞功能和特异性的主要调节者，抗TCR/CD3 单克隆抗体已在基础研究和临床中都被广泛应用。如何制备质量好、产量高的单克隆抗体是当前各有关实验室研究的重要问题。目前用于生产单克隆抗体的方法有2种：利用纯系小鼠生产免疫腹水法和杂交瘤细胞常规体外培养法［1,2］。鉴于本次试验样品备用于后续的研发，以及腹水法易保持生物学活性、耗时短和易操作等优点，故应用B淋巴细胞杂交瘤技术联合腹水法，制备并筛选了1株抗人CD3 单克隆抗体，并对其特性进行了鉴定和检测，获得较为满意的结果，现报道如下。 ??? 1? 材料与方法 ??? 1.1? 材料 ??? 1.1.1? 纯化抗原、抗体与